|
| Status |
Public on Jul 31, 2009 |
| Title |
D.rerio_embryo_CR_vs_E11_embryo_24hpiB |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
D.rerio_embryo_CR
|
| Organism |
Danio rerio |
| Characteristics |
reference: Common reference (CR) consisting of a mixture of samples from embryo infections
|
| Biomaterial provider |
Astrid van der Sar, VUMC
|
| Treatment protocol |
For the duration of bacterial injections embryos were kept under anaesthesia in egg water containing 0.02% buffered 3-aminobenzoic acid ethyl ester (tricaine, Sigma). Embryos were staged at 28 hours post fertilization (hpf) by morphological critera (Kimmel et al.) and approximately 50 cfu of E11 bacteria were injected into the caudal vein close to the urogenital opening. As a control an equal volume of PBS was likewise injected. Infection experiments were carried out in triplicate on separate days and pools of 15-20 embryos were taken at 2, 24 and 120 hours post infection (hpi).
|
| Growth protocol |
Embryos were grown at 28,5 -30 °C in egg water (60µg/ml Instant Ocean see salts).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos were directly homogenized in TRIZOL® Reagent. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit (Qiagen) according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies). Samples used for microarray analysis had an average RIN value of 9 and a minimum RIN value of 8.
|
| Label |
Cy3
|
| Label protocol |
Amino Allyl modified aRNA was synthesized in one amplification round from 1 μg of total RNA using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion). Subsequently, 6 ug of Amino Allyl modified aRNA was used for coupling of monoreactive Cy5 dye(GE Healthcare) and column purified.
|
| |
|
| Channel 2 |
| Source name |
D.rerio_embryo_E11_24hpi
|
| Organism |
Danio rerio |
| Characteristics |
tissue: 15-20 embryos
time: 24 hours post infection
infection: 50 cfu of E11 M. marinum strain
|
| Biomaterial provider |
Astrid van der Sar, VUMC
|
| Treatment protocol |
For the duration of bacterial injections embryos were kept under anaesthesia in egg water containing 0.02% buffered 3-aminobenzoic acid ethyl ester (tricaine, Sigma). Embryos were staged at 28 hours post fertilization (hpf) by morphological critera (Kimmel et al.) and approximately 50 cfu of E11 bacteria were injected into the caudal vein close to the urogenital opening. As a control an equal volume of PBS was likewise injected. Infection experiments were carried out in triplicate on separate days and pools of 15-20 embryos were taken at 2, 24 and 120 hours post infection (hpi).
|
| Growth protocol |
Embryos were grown at 28,5 -30 °C in egg water (60µg/ml Instant Ocean see salts).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos were directly homogenized in TRIZOL® Reagent. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit (Qiagen) according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies). Samples used for microarray analysis had an average RIN value of 9 and a minimum RIN value of 8.
|
| Label |
Cy5
|
| Label protocol |
Amino Allyl modified aRNA was synthesized in one amplification round from 1 μg of total RNA using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion). Subsequently, 6 ug of Amino Allyl modified aRNA was used for coupling of monoreactive Cy5 dye(GE Healthcare) and column purified.
|
| |
|
| |
| Hybridization protocol |
The dual colour hybridization of the microarray chips was performed at ServiceXS (ServiceXS, Leiden, The Netherlands) according to Agilent protocol G4140-90050 v.5.7 (www.Agilent.com) for Two-Color Microarray-Based Gene Expression Analysis.
|
| Scan protocol |
Microarray data were processed from raw data image files with Feature Extraction Software 9.5.3 (Agilent Technologies).
|
| Description |
CR_vs_E11_embryo_24hpiB
|
| Data processing |
Processed data were subsequently imported into Rosetta Resolver 7.1 (Rosetta Biosoftware, Seattle, Washington) and subjected to default ratio error modelling. Data were analyzed at the level of UniGene clusters (UniGene build #105). The significance cut-off for ratios of infected versus control was set at P ≤ 10-4.
|
| |
|
| Submission date |
Mar 19, 2009 |
| Last update date |
Jun 02, 2009 |
| Contact name |
Anna Magdalena Zakrzewska |
| E-mail(s) |
ania.zakrzewska@gmail.com
|
| Phone |
020-6923990
|
| Fax |
-
|
| URL |
http://-
|
| Organization name |
University of Leiden
|
| Department |
IBL
|
| Lab |
A. Meijer
|
| Street address |
Wassenaarseweg 64
|
| City |
Leiden |
| ZIP/Postal code |
2333 AL |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL7735 |
| Series (1) |
| GSE15328 |
Specificity of the zebrafish host transcriptome response to acute and chronic mycobacterial infection |
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